Advances in recombinant DNA technology have led to progress in the development of gene transfer between organisms. At this time, numerous efforts are being made to produce chemical, pharmaceutical, and biological products of economic and commercial interest through the use of gene transfer techniques.
One of the key elements in genetic manipulation of both prokaryotic and eukaryotic cells is the development of vectors and vector-host systems. In general, a vector is a nucleic acid molecule capable of replicating or expressing in a host cell. A vector-host system can be defined as a host cell that bears a vector and allows the genetic information it contains to be replicated and expressed.
Vectors have been developed from viruses with both DNA and RNA genomes. Viral vectors derived from DNA viruses that replicate in the nucleus of the host cell have the drawback of being able to integrate into the genome of said cell, so they are generally not very safe. In contrast, viral vectors derived from RNA viruses, which replicate in the cytoplasm of the host cell, are safer than those based on DNA viruses, since the replication occurs through RNA outside the nucleus. These vectors are thus very unlikely to integrate into the host cell's genome.
cDNA clones have been obtained from single-chain RNA viruses with positive-polarity [ssRNA(+)], for example, picornavirus (Racaniello & Baltimore, 1981); bromovirus (Ahlquist et al., 1984); alphavirus, a genus that includes the Sindbis virus; Semliki Forest virus (SFV) and the Venezuelan equine encephalitis virus (VEE) (Rice et al., 1987; Liljeström and Garoff, 1991; Frolov et al., 1996; Smerdou and Liljestrom, 1999); flavivirus and pestivirus (Rice and Strauss, 1981; Lai et al., 1991; Rice et al., 1989); and viruses of the Astroviridae family (Geigen-muller et al., 1997). Likewise, vectors for the expression of heterologous genes have been developed from clones of DNA complementary to the genome of ssRNA(+) virus, for example alphavirus, including the Sindbis virus, Semliki Forest virus (SFV), and the Venezuelan equine encephalitis (VEE) virus (Frolov et al., 1996; Liljeström, 1994; Pushko et al., 1997). However, all methods of preparing recombinant viruses starting from RNA viruses are still complicated by the fact that most of the viruses comprise sequences which are toxic for bacteria. Preparing a cDNA of the viral RNA and subcloning of the cDNA in bacteria therefore often leads to deletion or rearrangement of the DNA sequences in the bacterial host. For this purpose most of the commonly used subcloning and expression vectors cannot be used for preparation of large DNA sections derived from recombinant RNA viruses. However, obtaining vectors, which can carry long foreign DNA sequences is required for a number of aspects in the development of pharmaceuticals, specifically vaccines.
The coronaviruses are ssRNA(+) viruses that present the largest known genome for an RNA virus, with a length comprised between about 25 and 31 kilobases (kb) (Siddell, 1995; Lai & Cavanagh, 1997; Enjuanes et al., 1998). During infection by coronavirus, the genomic RNA (gRNA) replicates and a set of subgenomic RNAs (sgRNA) of positive and negative polarity is synthesized (Sethna et al., 1989; Sawicki and Sawicki, 1990; van der Most & Spaan, 1995). The synthesis of the sgRNAs is an RNA-dependent process that occurs in the cytoplasm of the infected cell, although its precise mechanism is still not exactly known.
The construction of cDNAs that code defective interfering (DI) genomes (deletion mutants that require the presence of a complementing virus for their replication and transcription) of some coronaviruses, such as the murine hepatitis virus (MHV), infectious bronchitis virus (IBV), bovine coronavirus (BCV) (Chang et al., 1994), and porcine gastroenteritis virus (TGEV) (Spanish Patent Application P9600620; Méndez et al., 1996; Izeta et al., 1999; Sánchez et al., 1999) has been described. However, the construction of a cDNA clone that codes a complete genome of a coronavirus has not been possible due to the large size of and the toxic sequences within the coronavirus genome.
In summary, although a large number of viral vectors have been developed to replicate and express heterologous nucleic acids in host cells, the majority of the known vectors for expression of heterologous genes are not well suited for subcloning of RNA viruses. Further, the viral vectors so obtained present drawbacks due to lack of species specificity and target organ or tissue limitation and to their limited capacity for cloning, which restricts the possibilities of use in both basic and applied research.
Hence there is a need for methods to develop new vectors for expression of heterologous genes that can overcome the aforesaid problems. In particular, it would be advantagous to have large vectors for expression of heterologous genes with a high level of safety and cloning capacity, which can be designed so that their species specificity and tropism can be controlled.